CN1471578A - Method of accumulating foreign gene product in plant seed at high level - Google Patents
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Abstract
The present inventors succeeded in developing a vector that expresses high levels of a foreign gene in plant seeds by utilizing a 5'-untranslated region of a gene encoding a seed storage protein. The inventors also succeeded in accumulating high levels of a foreign gene product in plant seeds by utilizing a seed storage protein defective mutant as a target for foreign gene transfer.
Description
Technical field
The present invention relates to the high-level method of accumulating exogenous genes products in plant seed.
Background technology
According to the solubleness of seed storage protein, it can be divided into four histones, i.e. gluten, sphaeroprotein, prolamine and white protein usually.Be different from other cereal such as wheat and corn, the storage protein of rice paddy seed mainly is a gluten, accounts for the 70-80% of seed storage protein.Glutenin gene group in each monoploid genome comprises about 10 genes, and these genes are divided into two classes, GluA and GluB, and they have the homology of 60-65% on amino acid sequence level in the coding region.Every class comprises that about 5 kinds have 80% or the group of higher homology on amino acid sequence level.Glutenin gene is expressed specifically in endosperm and is accumulated.The tissue specificity that gluten is expressed is subjected to very strict regulation and control, and gluten is not expressed in other tissue such as leaf and root.Except GluA-3, the expression of glutenin gene is normally regulated and control; Pattern below their mRNA level shows: (the 5th day) occurs after bloom 5 days, reaches maximum value in the time of about 15 days, and after reduction.The GluB-1 gene has the strongest promoter activity in the glutenin gene group.
Isolated such rice mutant, its gluten of accumulating is that the amount of main seed storage protein reduces.For example Iida etc. isolates following cryptic mutant from the rice varieties Koshihikari that gammairradiation is crossed, and this mutant lacks the acid subunit α 1 of gluten, one of α α 2 or α 3.These phenotypes are subjected to single recessive gene (being glu1, glu2 or glu3) regulation and control respectively.Hybridization by above-mentioned three kinds of mutant also can obtain α 1, the mutant plant that α 2 and α 3 lack (α 123) (Iida, S et al., Theor.Appl.Genet.94:177-183 (1997)).
LGC-1 (low glutelin content-1) is the mutant that is selected from the Nihonmasari that handles with EMS, and it has the phenotype (Iida, S.et al., Theor.Appl.Genet.87:374-378 (1993)) that gluten levels significantly lowers.Further identify the level that LGC-1 has prolamine and a sphaeroprotein and increase feature.LGC-1 is regulated and control by single dominant gene.By to LGC-1 and α 1, defective gene mapping in α 2 and α 3 deficient mutants shows that the glutenin gene (glu1) of the sudden change in protein gene (lgc-1) that suddenlys change and the mutant that lacks α 1 among the LGC-1 is positioned at identical seat.Use the result of the Southern hybridization that gluten (GluB) gene carries out as probe to show, LGC-1 comprises sudden change near GluB gene or its.LGC-1 and original kind Nihonmasari heading (head spout) GluB expression of gene level in the endosperm after about 16 days relatively, the analytical results of Northem trace shows that the expression of GluB in LGC-1 significantly reduces.
Glycinin in the known soybean is a kind of seed storage protein.Glycinin produces with the form of the precursor polypeptide of about 60kDa, signal peptide wherein, and Acid polypeptide and basic polypeptide combine; In the subsequent process, signal peptide is cut off.After this, form subunit, wherein two peptide species that produce because of the cutting in Asn-Gly site-be specificity Acid polypeptide (A) and basic polypeptide (B)-pass through disulfide linkage polymerization.Six such subunits are assembled into six aggressiveness, and are stored in the proteoplast (PB).This six aggressiveness are because the cause of its settling ratio (11S) also claims " 11S seed storage protein ".The glycinin subunit is divided into group I and group II based on the primary structure of its cDNA and the homology of its aminoacid sequence.The subunit A1aB1b of present known group I, subunit A3B4 and the A5A4B3 of A1bB2 and A2B1a and group II.Known 6 such subunits almost are to make up at random in the glycinin of soybean.And, it is reported that the Toplink derived from the A1aB1b subunit of the glycinin of soybean combines (Shio Makino with bile acide, The FoodIndustry 39 (24): 77-87 (1996)), this ability that shows cholesterol levels in the soybean protein reduction blood depends on the A1aB1b subunit.
Summary of the invention
The inventor concentrates the favourable physiological function of the glycinin of research soybean, effect such as above-mentioned reducing cholesterol, and successfully obtain a kind of paddy rice, the composition of storage protein changes (patent 3030339) by express the A1aB1b gene in the rice paddy seed endosperm in its seed.Yet,, need high-caliber expression in order to produce the physiological action of expection by edible this paddy rice.Therefore, need development and utilization can in paddy rice, accumulate the technology of high-level exogenous genes products.The present invention considers these demands and carries out, the purpose of this invention is to provide a kind of method of accumulating high-level exogenous genes products in plant seed.
In order to reach above purpose, the inventor tries hard to improve promotor with high level expression foreign gene in plant seed.By detect the promoter region of rice paddy seed storage protein gluten GluB-1 gene, demonstrate the conventional carrier that is used to express the glycinin gene do not comprise fully 5 of glutenin gene '-non-translational region.The inventor concentrate 5 of research glutenin gene '-non-translational region (not recognizing its importance as yet), and detect 5 '-non-translational region inserts the level of accumulating whether expression vector can improve mRNA.The result shows, compares with conventional glycinin gene transfer body, does not improve on expression level, and this transductant has inserted the enhancer sequence (pSaDb) of the photosynthetic gene of tobacco between GluB-1 gene promoter and glycinin gene (A1aB1b).Yet, gluten complete 5 '-insertion (ATG) of non-translational region significantly raise mRNA and the proteinic level of accumulating.
Previous research is from the maximum capacity of the genetic expression (transcribe and translate) that reckons without expression alien gene plant.Therefore, only try hard to foreign gene is imported the plant variety that is generally used for testing.The inventor concentrates " maximum capacity " of research plant, and supposition apparatus body protein deficient mutants can higher levels ofly be accumulated exogenous genes products.Therefore, the inventor attempts with the expression of plants that suddenlys change and accumulates foreign gene.
Several mutant that lack main storage protein in the present known paddy rice are such as LGC-1 and α 123.The inventor estimates, in this class seed storage protein deficient mutants, the quantity that can be used for the total free aminoacids of protein translation is higher than the quantity in the normal plants, because this class total free aminoacids is not used for the biosynthesizing of the storage protein normally accumulated.And, the inventor think the use of LGC-1 two-story valley protein promoter can high level expression foreign gene because the expression of glutenin gene is suppressed in LGC-1, and can uses thus and be used for the transcription factor that gluten expresses at first and come expression alien gene.Therefore, the inventor so that the glycinin gene is imported this mutant, detects the level of accumulating glycinin in its seed with LGC-1 or α 123 and glycinin transductant 11-5 hybridization.Found that with 11-5 and compare that the amount of the glycinin of being accumulated significantly raises in LGC * 11-5 and α 123 * 11-5 system.
Therefore 5 of the gene of the inventor by utilizing the coding seed storage protein '-non-translational region successfully developed a kind of in plant seed the carrier of high level expression foreign gene.The inventor is also by utilizing the seed storage protein deficient mutants as the target of transgenosis and successfully accumulate exogenous genes products in plant seed high-levelly, and finally draws the present invention thus.
More particularly, the invention provides:
(1) a kind of method of accumulating exogenous genes products in plant seed comprises following step: foreign gene is imported in the plant of endogenous seed storage protein defective, and in this plant expression alien gene;
(2) according to the method for (1), wherein to import what use be a kind of carrier to foreign gene, and this carrier comprises the foreign gene that is connected with the promotor downstream with handling, and described promotor guarantees that foreign gene expresses in plant seed;
(3) according to the method for (1), wherein the importing of foreign gene is by carrying out with the plant hybridization that comprises described foreign gene;
(4) according to the method for (2), 5 of the gene of the seed storage protein of wherein will encoding '-non-translational region is inserted into foreign gene and guarantees that foreign gene is in plant seed between expression promoter;
(5) according to the method for (4), wherein 5 '-non-translational region is complete;
(6) according to the method for (4) or (5), wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, in prolamine and the white protein group 5 of proteic gene '-non-translational region;
(7) according to the method for (6), wherein 5 '-non-translational region comprises the nucleotide sequence of SEQ ID NO:1;
(8) according to each described method in (1)-(7), wherein the defective type seed storage protein is selected from gluten in the plant, sphaeroprotein, prolamine and white protein;
(9) transformed plant cells of endogenous seed storage protein defective has wherein been led foreign gene;
(10) transformed plant cells of endogenous seed storage protein defective has wherein imported a kind of carrier that comprises the foreign gene that is connected with the promotor downstream with handling, and described promotor guarantees that foreign gene expresses in plant seed;
(11) according to the transformed plant cells of (10), 5 of the gene of the seed storage protein of wherein will encoding '-non-translational region is inserted into foreign gene and guarantees that foreign gene is in plant seed between expression promoter;
(12) according to the transformed plant cells of (11), wherein 5 '-non-translational region is complete;
(13) according to the transformed plant cells of (11) or (12), wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, and proteic gene 5 in prolamine and the white protein '-non-translational region;
(14) according to the transformed plant cells of (13), wherein 5 '-non-translational region comprises the nucleotide sequence of SEQ ID NO:1;
(15) according to each described transformed plant cells in (9)-(14), wherein the defective type seed storage protein is to be selected from gluten in the plant, sphaeroprotein, prolamine and white protein;
(16) a kind of transgenic plant, comprise (9)-(15) in each described transformed plant cells;
(17) a kind of carrier, comprise 5 of the gene of guaranteeing expression promoter and the coding seed storage protein that links to each other with this promotor in plant seed '-non-translational region;
(18) according to the carrier of (17), wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, and proteic gene 5 in prolamine and the white protein '-non-translational region;
(19) according to the carrier of (18), wherein 5 of glutenin gene '-non-translational region comprises the nucleotide sequence of SEQ ID NO:1;
(20), guarantee that wherein expression promoter is that coding is selected from gluten, sphaeroprotein, the promotor of proteic gene in prolamine and the white protein in plant seed according to each described carrier in (17)-(19);
(21) according to each described carrier in (17)-(20), wherein foreign gene and 5 '-downstream of non-translational region is connected with handling;
(22) transformed plant cells of the described carrier of a kind of leading (21);
(23) a kind of comprising (22) described transformed plant cells transgenic plant;
(24) a kind of is the offspring of conversion plant of (16) or (23) or clone's transgenic plant; With
(25) a kind of (16), the breeding material of each described transgenic plant in (23) and (24).
The invention provides a kind of method of in plant seed, accumulating high-level exogenous genes products.The method is characterized in that and utilize the target of endogenous seed storage protein deficient mutants plant as expression alien gene.Term " defective type " comprises that not only complete defective also comprises the part defective herein.In described plant, the quantity that can be used for the total free aminoacids of protein translation is considered to be higher than in the normal plants, and this causes accumulating effectively the translation product of foreign gene in seed.Defective type seed storage protein in the plant there is not special restriction; The present invention includes, for example, gluten, sphaeroprotein, prolamine and white protein.
These protein deficient plants can be selected from the plant seed that radiation such as gamma-radiation was handled, or the plant seed that suddenlys change and induce reagent such as EMS and MNU to handle.The mutant plant can pass through seed profile method (bisection method) screening.Concrete is, seed is divided into two, and extracts protein has desired phenotype with screening seed in endosperm.The offspring can derive from the embryo corresponding to the selected endosperm with desired phenotype.
Perhaps, the plant with seed storage protein of accumulating the level reduction can suppress or the generation of antisense method by being total to.For suppressing altogether, a part is modified in the gene of the seed storage protein that needs are reduced, and it is imported plant.In this mode, be higher than the gene of certain value with respect to the homology of this modifying factor, its expression (for example is suppressed, in above-mentioned LGC-1 mutant, gluten α 1 subunit gene is undergone mutation, and prompting has produced common inhibition, and this mutant comes from the plant of shining with gamma-radiation).On the other hand, in the antisense method, with the DNA importing plant of encoding antisense RNA, this RNA and the gene transcription product complementation that will reduce.
According to the present invention, can also use the known rice mutant that lacks main storage protein, such as LGC-1 and α 123.
Any gene that is adapted at expressing in the plant seed all can be used as foreign gene.For example, can use the soybean glycinin to produce as foreign gene has than high additive value, is rich in nutrition, has fabulous machining feature and/or can keeps and improve healthy crop (patent 3030339) by reducing people's blood cholesterol levels.Perhaps, the vaccine gene of passive immunotherapy, the glutenin gene of modification (wherein physiologically active peptide having been mixed its variable region) or useful enzyme gene can be imported paddy rice, to produce the paddy rice of high additive value.
For expression alien gene in plant seed, the preferential carrier that comprises foreign gene that uses, this foreign gene operably with guarantee that the downstream of expression promoter links to each other in plant seed.Phrase " operably connect " is meant that foreign gene links to each other with promotor herein, causes replying the activation of promotor and expression alien gene.
For example, in order to express in rice paddy seed, glutenin gene promotor (Takaiwa, F.et al., PlantMol.Biol.17:875-885 (1991)) can be used as the exogenous gene expression promotor.Leguminous plants, such as French beans (string bean), broad bean, pea; Or at oil seed plant such as peanut, sesame, Semen Brassicae campestris, cottonseed, Sunflower Receptacle when expressing in the and Flos Carthami, can be used the promotor of glycinin gene promoter or every kind of plant main storage protein gene separately.For example, Kidney bean protein gene promoter (Murai, N.et al., Science 222:476-482 (1983)) and cruciferin gene promoter (Rodin, J.et al., Plant Mol.Biol.20:559-563 (1992)) is respectively applied for French beans and Semen Brassicae campestris.Above-mentioned promotor of giving only is an example, can also use the promotor that is used for constitutive expression such as 35S promoter.
In order in plant seed, to accumulate exogenous genes products effectively, preferably in carrier, insert between promotor and foreign gene the coding seed storage protein gene 5 '-non-translational region.Described 5 '-example of non-translational region comprise 5 of those following proteic genes of encoding '-non-translational region: glutenin gene (X54313, the gluten GluA-3 gene of rice (Oryza sativa), gi|20207|emb|X54313.1|OSGLUA3[20207]; X54314, the gluten GluB-1 gene of rice, gi|20209|emb|X54314.1|OSGLUB1[20209]), sphaeroprotein (X62091, the lower molecular weight sphaeroprotein, gi|5777591|emb|X62091.1|OSLMWG[5777591]), prolamine (D11385, the prolamine mRNA of rice, complete cds, gi|218186|dbj|D11385.1|RICPLM[218186]), and white protein (D11431, the RA17 gene of paddy rice allergen protein, complete cds, gi|218194|dbj|D11431.1|RICRA17[218194]; D11432, the RA14 gene of paddy rice allergen protein, complete cds, gi|218192|dbj|D11432.1|RICRA14[218192]).Particularly preferably be 5 of complete form '-non-translational region.Also can use among the present invention derived from the coding two kinds of different seed storage proteins gene mosaic type 5 '-non-translational region.The GluB-1 gene complete 5 '-non-translational region shows in SEQ IDNO:1.
Use the gene manipulation techniques known to those skilled in the art to make up two kinds of carriers, a kind of be wherein 5 '-non-translational region is inserted into the carrier of guaranteeing expression promoter downstream in plant seed, a kind of carrier that wherein further inserts foreign gene.
With regard to essence of the present invention, in order to the source plant of the vegetable cell that imports carrier and unrestricted, so long as spermatophyte gets final product.For example, plant of the present invention comprises cereal, paddy rice for example, barley, wheat, rye and corn; Pulse family, French beans for example, broad bean and pea; With oil seed plant peanut for example, sesame, Semen Brassicae campestris, cottonseed, Sunflower Receptacle, and Flos Carthami etc.
Comprise the renewable various forms of plant that is in order to the vegetable cell form that imports carrier among the present invention.For example, comprise that form in the present invention has cultured cells, protoplastis, the seedling original hase, the polygerm body, hairly root and callus, but be not limited to these.Vegetable cell of the present invention also comprises the cell in the plant.
Can use the method known to those skilled in the art that carrier is imported vegetable cell.For example, these methods comprise use Agrobacterium tumefaciems or Agrobacterium rhizogenes transduce indirectly (Hiei, Y.et al., Plant be (1994) J.6:271-282; Takaiwa, F.et al., Plant Sci.111:39-49 (1995)); With direct transduction, representative is electroporation method (Tada, Y.et al., Theor.Appl.Genet.80:475 (1990)), polyoxyethylene glycol method (Datta, S.K.et al., Plant Mol.Biol.20:619-629 (1992)), and the method for particle bombardment (Christou, P.et al., Plant be (1992) J.2:275-281; Fromm, M.E., Bio/Technology 8:833-839 (1990)).
By making plant transformed cell regeneration can produce plant.The regenerated method is different according to the type of plant.Yet representational method comprises method (Fujimura, the T.et al. of Fujimura etc., PlantTissue Culture Lett.2:74 (1995)), the method of Armstrong etc. (Armstrong, C.L andPhillips R.L., Crop Sci.28:363-369 (1988)), and Radke, S.E. the method that waits (Radke, S.E.et al., Theor.Appl.genet.75:685-694 (1988)), correspond respectively to paddy rice, corn and Semen Brassicae campestris.
Except aforesaid method, can utilize hybridization that foreign gene is imported endogenous seed storage protein deficient plants.For example, at first, the plant of carrying foreign gene in the genome produces by above-mentioned carrier is imported.Then, the hybridization of this plant and endogenous seed storage protein deficient plants is to import foreign gene endogenous seed storage protein deficient plants.
In case obtain just can obtain the plant offspring by sexual propagation wherein with the transgenic plant of foreign gene quiding gene group.Perhaps, can obtain reproductive material (for example seed, plant, callus and protoplastis) from plant and its offspring or its clone, with it as parent material, in order to produce a large amount of plants.By expression alien gene, transgenic plant of the present invention can be accumulated high-caliber exogenous genes products in seed.Therefore, according to the feature of the concrete exogenous genes products of accumulating in the seed, the edibleness of seed (fbod value), machining feature, sanatory effects etc. can be changed effectively.And, by in seed, accumulating antibody or enzyme, can prepare pharmaceutical product and Industrial materials effectively.
The accompanying drawing summary
Fig. 1 for be used to detect 5 '-construct of the effect of non-translational region (UTR).
Fig. 2 accumulates the comparative result of level for the glycinin of being surveyed in the plant, and this plant transforms with the construct that contains 5 of Fig. 1 '-non-translational region.
Fig. 3 accumulates and expresses for soybean glycinin in transgenic paddy rice seed.(A) for proving that SDS-PAGE analyzes (last figure) and Northern engram analysis (figure below) result's photo.(B) for showing the quantitative of (A) middle result and comparing.N represents to comprise the plant of gluten and glycinin non-translational region chimeric sequences; ATG represent to comprise glutenin gene complete 5 '-non-translational region; 11-5 represents conventional glycinin gene transfer body; Non-tra is a non-transgenic plant.
The photo of Fig. 4 is the SDS-PAGE analysis influence that show, that gluten defective type phenotype is accumulated exogenous genes products by endosperm protein.11-5 is for comprising the transgenosis Matsuyama-mii of glycinin (A1aB1b) gene; LGC refers to LGC-1; Non-tra is a non-transgenic plant.
Embodiment
To use embodiment to specifically describe the present invention below, but this is not inferred as limitation of the present invention.
(embodiment 1)
Utilize improved promotor to make up soybean glycinin expression vector and generation table Reach the rice plants of soybean glycinin
(1)
The structure of mosaic gene and transgenosis
The cDNA of glycinin (A1aB1b) of will encoding links to each other with the GluB-1 gene promoter.Between this cDNA and promotor, insert the chimeric sequences (45bp) (for N) of gluten non-translational region (+1 to 18) and glycinin (27 to ATG) non-translational region, or insertion GluB-1 gene is complete 5 '-non-translational region (for ATG) is (Fig. 1).In contrast, make up a kind of expression vector, it has inserted 5 of the photosynthetic gene translation enhancer sequence of tobacco pSaDb '-non-translational region.These plasmids that utilize the edaphic bacillus method will comprise these mosaic genes import paddy rice (Oryza sativa cv kitaake) (Goto, F.et al., Nat.Biotechnol.17:282-286 (1999)).
(Oryza sativa cv Mateuyama-mii) selects 11-5 from paddy rice, and the cDNA of the glycinin (A1aB1b) of wherein encoding shifts by electroporation method with the mosaic gene that GluB-1 gene promoter (1302 to+18) links to each other.
(2)
5 of GluB-1 gene '-non-translational region is to the influence of exogenous gene expression in the plant seed
, with gene transfered plant the protein level of gained plant seed (T1) is analyzed by the edaphic bacillus method.N, the relatively demonstration of pSaDb and ATG, it is higher to accumulate the frequency that the plant of high-level glycinin occurs, order following ATG＞N＞pSaDb (Fig. 2).
Then, from the N that accumulates high-level glycinin and ATG transfer-gen plant, select plant respectively, make its selfing with the screening homozygote with high expression level.Then, mRNA and the protein level in the following analytical pure zygote.In RNA analyzes, at first extract RNA by the SDS-phenol method.About 15 days 12 the immature seed liquid nitrogen freezings in back of blooming grind to form tiny powder in the mortar.Make it to mix, extract total nucleic acid with damping fluid (0.1M Tris-HCl (pH9.0), 1%SDS, 0.1M NaCl, 5mM EDTA) and phenol-chloroform-primary isoamyl alcohol (25: 24: 1).Centrifugal sample reclaims supernatant, and uses phenol-chloroform-primary isoamyl alcohol (25: 24: 1) to extract once more.Collect total nucleic acid by ethanol precipitation, heavily be dissolved in the distilled water.Then, precipitated rna in 2M LiCl.By centrifugal recovery as the RNA sample.With RNA electrophoresis on 1.2% sepharose, and transfer on the nylon membrane.42 ℃ of prepared films are in 50% (v/v) methane amide, 6 * SSC, in 0.5% (w/v) SDS and the 5 * Denhardt solution with
32The glycinin of p-mark (A1aB1b) cDNA hybridization.Then, with film in room temperature in 2 * SSC, 0.1%SDS solution is given a baby a bath on the third day after its birth time, then 55 ℃ in 0.1 * SSC, flushing is 20 minutes in the 0.1%SDS solution.In protein analysis, total protein uses 250ul to extract damping fluid (62.5mMTris-HCl (pH6.8) comprises 10% (v/v) glycerine, 0.25%w/v) SDS and 5%2-mercaptoethanol) by every 10mg mature seed.The protein that is extracted was handled 5 minutes at 100 ℃, carried out SDS-PAGE then.SDS-PAGE carries out (acrylamide: N, N ' methylene bisacrylamide=30: 0.8) with 1 5% (w/v) polyacrylamide gel.
As a result, the expression level of finding A1aB1b among N and the ATG is respectively among the 11-5 1.43 times and 6.56 times (Fig. 3).Separate the acid subunit of glycinin by SDS-PAGE, accumulate level with comparison protein, found that, A1aB1b accumulates level and is respectively among the 11-5 1.40 times and 1.62 times (Fig. 3) among N and the ATG.These results show, 5 of GluB-1 gene '-non-translational region particularly completely 5 '-non-translational region be inserted in the GluB-1 gene promoter and the cDNA of the glycinin (A1aB1b) of encoding between, can improve the expression of exogenous gene level effectively.
(embodiment 2)
Utilize mutant exploitation high level to accumulate the technology of exogenous genes products
11-5 (Momma, K.et al., Biosci.Biotechnol.Biochem.63:314-318 (1999)) with LGC-1 (Iida, S.et al., Theor.Appl.Genet.87:314-378 (1993)) or α 123 (Iida, S.etal., Theor.Appl.Genet.94:177-183 (1997)) hybridization, collect their F1 seed, seed is divided into two (seed profile method) extracted and analyzed by SDS-PAGE with endosperm protein.Based on the analytical results of SDS-PAGE, show corresponding to the strong band of glycinin with corresponding to the seed of the weak band of the acid subunit of gluten is screened.Repeat such screening, obtain all plants of homogeneous of all phenotypes.
By the endosperm protein (Fig. 4) among SDS-PAGE analysis LGC * 11-5 and the α 123 * 11-5.As a result, LGC * 11-5 shows the phenotype of LGC-1, wherein corresponding to the band of the acid subunit of glycinin of all 37-39kDa die down (total amount of gluten reduces to about 1/3rd).On the contrary, 11-5 compares with the glycinin transductant, corresponding to the remarkable grow of band (1.4 times) of the acid subunit of transgene product gluten.On the other hand, α 123 * 11-5 defective at the acid subunit α 1 of gluten, α 2 and α 3, and the identical phenotype of demonstration and α 123.In α 123 * 11-5,11-5 compares with the glycinin transductant, corresponding to the remarkable grow of band (1.7 times) of the acid subunit of transgene product glycinin.
Then, quantitatively accumulate the amount of transgene product glycinin A1aB1b.Concrete is, extracts total protein in seed, and point utilizes anti--glycinin (A1aB1b) antibody to carry out immunoblotting on nitrocellulose filter.As a result, with the plant seed of LGC-1 hybridization in, the band of the acid subunit of transgene product glycinin significantly strengthens.And, to those of α 123 hybridization in obtain similar result.These results show, seed storage protein defective type phenotype is increased to can accumulates in seed endosperm in the strain of exogenous genes products, can accumulate exogenous genes products high-levelly.
Commercial Application
The invention provides a kind of in the plant seed high level accumulate the method for exogenous genes products. The present invention Method can be used as important basic technology and be used for developing useful agricultural products and food.
Sequence table＜110〉Japan as Represented by Director General of Ministry of Agriculture, Forestry an (National Institute of Agrobiological Sciences)
Biological department specific industry technical research promotion institution (Bio-oriented Technology ResearchAdvancement Institution)＜120〉in vegetable seeds high level accumulate the method for exogenous genes products＜130〉MOA-A0004P＜140＜141＜150〉JP 2000-251606＜151 2000-08-22＜160 1＜170〉PatentIn Ver.2.0＜210 1＜211〉44＜212〉DNA＜213〉rice (Oryza sativa (Kasalath))＜400〉1tcacatcaat tagcttaagt ttccataagc aagtacaaat agct 44
Claims (25)
1. method of accumulating exogenous genes products in plant seed comprises following step: foreign gene is imported in the plant of the endogenous seed storage protein of defective, and in this plant expression alien gene.
2. the process of claim 1 wherein that foreign gene uses carrier to import, this carrier comprises the foreign gene that is connected with the promotor downstream with handling, and described promotor can guarantee that foreign gene expresses in plant seed.
3. the process of claim 1 wherein that the importing of foreign gene is by carrying out with the hybridization of the plant that comprises described foreign gene.
4. the method for claim 2,5 of the gene of the seed storage protein of wherein will encoding '-non-translational region is inserted into foreign gene and guarantees that foreign gene is in plant seed between expression promoter.
5. the method for claim 4, wherein 5 '-non-translational region is complete.
6. claim 4 or 5 method, wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, in prolamine and the white protein group 5 of proteic gene '-non-translational region.
7. the method for claim 6, wherein 5 '-non-translational region comprises the nucleotide sequence of SEQ ID NO:1.
8. each described method among the claim 1-7, wherein the defective type seed storage protein is selected from gluten in the plant, sphaeroprotein, prolamine and white protein.
9. the transformed plant cells of the endogenous seed storage protein of defective has wherein been led foreign gene.
10. the transformed plant cells of the endogenous seed storage protein of defective has wherein imported a kind of carrier that comprises the foreign gene that is connected with the promotor downstream with handling, and described promotor guarantees that foreign gene expresses in plant seed.
11. the transformed plant cells of claim 10,5 of the gene of the seed storage protein of wherein will encoding '-non-translational region is inserted in the expression vector foreign gene and guarantees that foreign gene is in plant seed between expression promoter.
12. the transformed plant cells of claim 11, wherein 5 '-non-translational region is complete.
13. the transformed plant cells of claim 11 or 12, wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, and proteic gene 5 in prolamine and the white protein group '-non-translational region.
14. the transformed plant cells of claim 13, wherein 5 '-non-translational region comprises the nucleotide sequence of SEQ ID NO:1.
15. each described transformed plant cells among the claim 9-14, wherein the defective type seed storage protein is selected from gluten in the plant, sphaeroprotein, prolamine and white protein.
16. transgenic plant comprise each described transformed plant cells among the claim 9-15.
17. a carrier, comprise guarantee in plant seed expression promoter with link to each other with this promotor and the gene of the seed storage protein of encoding complete 5 '-non-translational region.
18. the carrier of claim 17, wherein 5 '-non-translational region is that coding is selected from gluten, sphaeroprotein, and proteic gene 5 in prolamine and the white protein '-non-translational region.
19. the carrier of claim 18, wherein 5 of glutenin gene '-non-translational region comprises the nucleotide sequence of SEQ IDNO:1.
20. each described carrier among the claim 17-19 guarantees that wherein expression promoter is that coding is selected from gluten, sphaeroprotein, the promotor of proteic gene in prolamine and the white protein in plant seed.
21. each described carrier among the claim 17-20, wherein foreign gene and 5 '-downstream of non-translational region is connected with handling.
22. a transformed plant cells has wherein imported the described carrier of claim 21.
23. transgenic plant that comprise the described transformed plant cells of claim 22.
24. transgenic plant, it is offspring or the clone of claim 16 or 23 described conversion plants.
25. the breeding material of each described transgenic plant in the claim 16,23 and 24.
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| JP251606/2000 | 2000-08-22 | ||
| JP2000251606A JP4565231B2 (en) | 2000-08-22 | 2000-08-22 | Method for highly accumulating foreign gene products in plant seeds |
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| CN1471578A true CN1471578A (en) | 2004-01-28 |
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| US (1) | US7473825B2 (en) |
| EP (1) | EP1312672B1 (en) |
| JP (1) | JP4565231B2 (en) |
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| CN (1) | CN1471578A (en) |
| AU (2) | AU7876501A (en) |
| CA (1) | CA2419282C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486851A (en) * | 2018-10-12 | 2019-03-19 | 武汉大学 | A method of improving recombinant protein expression in endosperm bioreactor |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7728192B2 (en) * | 1999-12-10 | 2010-06-01 | Kimmo Koivu | Process for converting storage reserves of dicotyledonous seeds into compositions comprising one or more gene products |
| AU2003289283B2 (en) | 2002-12-20 | 2008-05-08 | Incorporated Administrative Agency National Agriculture And Bio-Oriented Research Organization | Plant with reduced protein content in seed, method of constructing the same and method of using the same |
| US7847063B2 (en) | 2003-03-28 | 2010-12-07 | Sanwa Kagaku Kenkyusho Co., Ltd. | GLP-1 derivative |
| EP1668137B8 (en) * | 2003-08-27 | 2012-03-21 | ORF Liftaekni HF. | Enhancing accumulation of heterologous polypeptides in plant seeds through targeted suppression of endogenous storage proteins |
| JP4019147B2 (en) | 2003-10-31 | 2007-12-12 | 独立行政法人農業生物資源研究所 | Seed-specific promoter and its use |
| EP1808491A1 (en) * | 2006-01-13 | 2007-07-18 | Meristem Therapeutics S.A. | Genetically modified plant with modified storage protein content |
| JP5158639B2 (en) | 2008-04-11 | 2013-03-06 | 独立行政法人農業生物資源研究所 | Genes specifically expressed in the endosperm of plants, promoters of the genes, and use thereof |
| US20100313307A1 (en) * | 2008-06-28 | 2010-12-09 | Donald Danforth Plant Science Center | Protein production and storage in plants |
| KR101383340B1 (en) * | 2012-06-15 | 2014-04-10 | 건국대학교 산학협력단 | Method for Controling Growth and Development in the Arabidopsis Shoot System |
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| AU2811889A (en) * | 1987-10-20 | 1989-05-23 | Plant Genetic Systems N.V. | A process for the production of biologically active peptide via the expression of modified storage seed protein genes in transgenic plants |
| CA2092069A1 (en) * | 1992-03-27 | 1993-09-28 | Asako Iida | An expression plasmid for seeds |
| CA2158018C (en) * | 1993-03-02 | 2005-12-27 | Christine Irmgard Wandelt | Improved feedcrops enriched in sulfur amino acids and methods for improvement |
| JPH07213185A (en) | 1994-02-03 | 1995-08-15 | Sumitomo Chem Co Ltd | Plant improved in content of amino acid containing sulfur and method for improvement |
| DE19601365A1 (en) * | 1996-01-16 | 1997-07-17 | Planttec Biotechnologie Gmbh | Nucleic acid molecules from plants encoding enzymes involved in starch synthesis |
| IL127246A0 (en) | 1996-05-29 | 1999-09-22 | Hoechst Schering Agrevo Gmbh | Nucleic acid molecules encoding enzymes from wheat which are involved in starch synthesis |
| DE19653176A1 (en) | 1996-12-19 | 1998-06-25 | Planttec Biotechnologie Gmbh | New maize nucleic acid molecules and their use to produce a modified starch |
| JP3030339B2 (en) * | 1998-08-07 | 2000-04-10 | 農林水産省農業生物資源研究所長 | Transgenic plants expressing soybean glycinin |
| JP2002529079A (en) | 1998-11-10 | 2002-09-10 | マキシジェン， インコーポレイテッド | Modified ribulose 1,5-bisphosphate carboxylase / oxygenase |
| US6235976B1 (en) * | 1999-01-29 | 2001-05-22 | Pioneer Hi-Bred International, Inc. | Soybean variety 94B53 |
| US7417178B2 (en) * | 2000-05-02 | 2008-08-26 | Ventria Bioscience | Expression of human milk proteins in transgenic plants |
| US7741533B2 (en) * | 2000-11-07 | 2010-06-22 | Pioneer Hi-Bred International, Inc. | Grain quality through altered expression of seed proteins |
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- 2001-08-17 AU AU7876501A patent/AU7876501A/en active Pending
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- 2001-08-17 CN CNA018177891A patent/CN1471578A/en active Pending
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- 2001-08-17 KR KR10-2003-7002572A patent/KR20030028580A/en not_active Application Discontinuation
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486851A (en) * | 2018-10-12 | 2019-03-19 | 武汉大学 | A method of improving recombinant protein expression in endosperm bioreactor |
| WO2020074002A1 (en) * | 2018-10-12 | 2020-04-16 | 武汉大学 | Method for improving expression level of recombinant protein in endosperm bioreactor |
| CN109486851B (en) * | 2018-10-12 | 2022-04-01 | 武汉禾元生物科技股份有限公司 | Method for improving expression level of recombinant protein in endosperm bioreactor |
| CN114634559A (en) * | 2018-10-12 | 2022-06-17 | 武汉禾元生物科技股份有限公司 | Method for improving expression level of recombinant protein in endosperm bioreactor |
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| CA2419282A1 (en) | 2003-02-11 |
| CA2419282C (en) | 2012-10-16 |
| AU2001278765B2 (en) | 2005-08-18 |
| US7473825B2 (en) | 2009-01-06 |
| EP1312672B1 (en) | 2012-11-21 |
| US20040031075A1 (en) | 2004-02-12 |
| JP2002058492A (en) | 2002-02-26 |
| JP4565231B2 (en) | 2010-10-20 |
| ES2400135T3 (en) | 2013-04-05 |
| WO2002016604A1 (en) | 2002-02-28 |
| KR20030028580A (en) | 2003-04-08 |
| EP1312672A1 (en) | 2003-05-21 |
| EP1312672A4 (en) | 2006-06-28 |
| AU7876501A (en) | 2002-03-04 |
| AU2001278765C1 (en) | 2006-05-11 |
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